Inositol 1,4,5-trisphosphate (InsP3) mediates the release of Ca from intracellular stores by interacting with a family of highly conserved receptors which contain an intrinsic Ca release channel. The InsP3 receptor (InsP3R) shares both structural and sequence similarities with the ryanodine receptor (RyR), the primary Ca release channel of the sarcoplasmic reticulum (SR) in cardiac and skeletal muscle. The type-2 InsP3R is the predominant isoform expressed in cardiac myocytes, but its localization and function in heart have been elusive. In ventricular myocytes the InsP3R2 is targeted to the nuclear envelope and it interacts with Ca-calmodulin dependent protein kinase II (CaMKII). A goal of this project is to identify and characterize proteins interacting with the InsP3R2 expressed in cardiac myocytes that are critical for function and modulation of local Ca signaling events (regarding both regulation of InsP3R by CaMKII and regulation of CaMKII by Ca release from InsP3R). The central focus encompasses detailed study of InsP3R2/CaMKIIdelta interactions and the functional consequences with respect to Ca signaling and nuclear signaling in hypertrophy & heart failure. A second emphasis is the characterization, use and further development of novel FRET-based biosensors for in situ measurement of InsP3 liberation and concentration. There are 5 Specific Aims in this project that will address the InsP3R2/CaMKII interaction and function. 1) Test the hypothesis that the InsP3R2 localized to the nuclear envelope of cardiac myocytes interacts with CaMKII and forms a macromolecular complex. 2) Test the hypothesis that the interaction of CaMKII directly modulates the InsP3R2 Ca release channel properties. 3) Determine the physiological consequences of perturbing or disrupting the InsP3R2-CaMKII interaction. 4) Test the hypothesis that the spatial/temporal patterns of expression levels for the InsP3Rs are altered during ES cell development, hypertrophy and heart failure. 5) Generate a biological sensors to measure the free concentration of InsP3 in an intact cells. These studies will provide considerable new information regarding InsP3R in cardiac myocytes in health and disease. This is especially the case with respect to localization, molecular partners and regulation of function. Additionally, we anticipate that the InsP3-biosensors will become important new tools in the InsP3 field. This work is highly synergistic with all three other projects in the Program Project Grant.